Optical system and method for exciting and measuring fluorescence on or in samples treated with fluorescent

ABSTRACT

An optical system and method of exciting and measuring fluorescence on or in samples treated using fluorescent pigments using such an optical system having at least one first laser ( 1 ); a mirror ( 4 ); a deflection element ( 7 ); an optic ( 8 ); and a unit ( 10 ), which includes mirror ( 4 ) and optic ( 8 ) mounted fixed in relation to one another. The unit ( 10 ) is positioned so it is linearly movable back and forth along the optical axis ( 5 ) and is mechanically connected to an oscillating linear drive ( 11 ). The optic ( 8 ) additionally acts as a collimator and the mirror ( 4 ) additionally acts to deflect the collimated light ( 12 ) parallel to the optical axis ( 5 ).

RELATED APPLICATION DATA

This application claims priority of the Swiss Application No. CH 0144/01filed on Jan. 26, 2001 and of the International Application No.PCT/CH02/00014 filed on Jan. 10, 2002 of which the entire disclosure isincorporated herein by reference in both cases.

The present invention relates to an optical system for exciting andmeasuring fluorescence on or in samples treated with fluorescentpigments according to the preamble of at least one of the attachedindependent claims. Such optical systems are known, for example, asscanning light microscopes.

Scanning light microscopes have been known for several decades. Theirfunctional principal is based on a light beam being concentrated to asmall point of light (the first focal point) on a sample. The sample andthis point of light are mutually moved in such a way that a specificarea of the sample is scanned (rasterized) by the point of light. Thelight which penetrates the sample or is reflected by it and/or thefluorescence triggered on or in the sample during the scanning istherefore referred to as “light originating from the sample” and ismeasured by one or more photodetectors. An enlarged image is produced inthat an original measurement point is assigned a specific area on animage of the sample. In principle, such a scanning light microscopetherefore includes:

-   -   a light source, which produces a light beam;    -   a sample holder for holding the sample;    -   an optic for producing a first focal point on the sample;    -   an optical arrangement for imaging a second focal point using        the light which shines through the sample and/or is reflected by        the sample and/or which represents fluorescence triggered on or        in the sample;    -   a photodetector for measuring the intensity of the second focal        point; and    -   a scanning mechanism for mutual movement of the sample and first        focal point.

In a conventional scanning light microscope, the light beam is deflectedin the direction of the two spatial axes X and Y to illuminate thesample. This procedure hides the disadvantage that the angle ofincidence on the sample of the light refracted by the projective lensvaries and produces an aberration in the imaging of the sample light bythe objective lens. This aberration may be corrected through anappropriate construction of the objective lens. Such a lensdisadvantageously makes the optic more costly and simultaneously has alimiting effect in regard to the light collecting efficiency andoperating distance.

According to U.S. Pat. No. 5,081,350, this problem is solved in that adevice is disclosed therein using which the sample is scanned by a lightbeam. In this case, the device for illuminating the sample and thedevice for measuring the signal coming from the sample are mounted on aunit which is movable back and forth. The sample is mounted on a sampletable movable perpendicularly to this oscillation in this case, so thatscanning of the sample is possible with a constant angle of incidence ofthe illumination. Because, especially for the application of a rapidscanning method, the light source is preferably to be positioned outsidethe movable part of the scanning light microscope, in this case the useof glass fiber waveguides is suggested, which optically connect thelight source to the projective. However, there is the danger that thisglass fiber cable may be damaged by the frequent and rapid back andforth movement.

An improved device according to the species is known from U.S. Pat. No.5,260,569, which solves the problems of the related art described abovein that a scanning light microscope is suggested therein which, as alight source, includes a laser, a mirror for deflecting the light,coming out of the laser and incident parallel to an optical axis on themirror, in the direction of a sample, a deflection element fordeflecting this light onto this mirror, an optic for producing a firstfocal point, a unit, including the mirror and the optic, in which themirror and optic are positioned fixed in relation to one another, whichis linearly movable back and forth along the optical axis, anoscillating linear drive which is mechanically connected to this unit, asample table, movable at least in the direction of the X and Z spatialaxes, for aligning the sample in relation to the first focal point, anoptical arrangement for imaging a second focal point using the lightoriginating from the sample, an aperture plate, positioned in the secondfocal point, for masking light originating from the sample which meetsthis aperture plate at a distance greater than a specific distance, aspectral filter for selecting a component of the light originating fromthe sample which passes through the aperture plate, and a detector formeasuring the intensity of the light originating from the sample whichpasses through the aperture plate and is selected by the spectralfilter. The optic is additionally implemented as a collimator for thelight originating from the sample and the mirror is additionallyimplemented for deflecting this collimated light diametrically oppositeto the direction of incidence of the light from the laser and parallelto the optical axis.

In addition to all features of the preamble of at least one of theattached independent claims, U.S. Pat. No. 5,260,569 also discloses ascanning light microscope in which the light emitted by a light sourceis aligned in parallel using a collimating lens acting as a part of theprojective. The collimated light propagates in the direction parallel tothe scanning direction of the microscope. Therefore, the collimatedlight beam—independently of the actual position of the unit movable backand forth—is always incident from the same direction on the mirror whichis fixed in the unit movable back and forth. This has the consequencethat the light beam is always reflected by the mirror onto the sample inthe same direction and in collimated form. This collimated light is,after a 90. degree. reflection on the mirror, bundled into a first focalpoint using a further projective lens which is also fixed in the unitmovable back and forth. Therefore, the scanning or rasterizing of thesample may be performed using the unit movable back and forth and usingthe light of a light source which is attached to the unit movable backand forth. However, the attachment of the light source and photodetectoroutside the unit movable back and forth is preferable, so that this unitmay be made simpler and lighter—to allow more rapid scanning.

The object of the present invention is to suggest an alternative opticalsystem and/or an alternative optical method which opens up additionalpossibilities for a simpler, more flexible system construction and/orsystem use and essentially has the advantages of the related art.

According to a first aspect, this object is achieved by a systemcorresponding to the combination of features as set forth in at leastone of the attached independent claims, in which, in addition to thefeatures of the preamble known from the most similar related or closestprior art, it is suggested that the optical system include a continuousgeometrical axis G, which does not run through the sample, on which theoptical arrangement, the second focal point, the aperture plate, thespectral filter, and the detector—together with the mirror and thedeflection element—are positioned, this geometrical axis G being atleast partially identical to the optical axis in the region between themirror and the detector. Further features according to the presentinvention result from the dependent claims.

According to a second aspect, this object is achieved by a methodcorresponding to the combination of features as set forth in at leastone of the attached independent claims, in which, in addition to thefeatures of the preamble known from the most similar related or closestprior art, it is suggested that an optical system be provided having acontinuous geometrical axis G which does not run through the sample, onwhich the optical arrangement, the second focal point, the apertureplate, the spectral filter, and the detector—together with the mirrorand the deflection element—are positioned, this geometrical axis G beingat least partially identical to the optical axis in the region betweenthe mirror and the detector. Further features according to the presentinvention result from the dependent claims.

The present invention will now be described in greater detail on thebasis of schematic illustrations, which merely represent preferredexamples of embodiments and do not restrict the scope of the disclosurein regard to the present invention.

FIG. 1 shows a quasi-spatial schematic illustration of an optical systemaccording to a first embodiment having two lasers;

FIG. 2 shows a vertical partial section of an optical system accordingto a second embodiment having at least one laser;

FIG. 3 shows a vertical partial section of an optical system accordingto a third embodiment having at least one laser.

FIG. 1 shows a quasi-spatial schematic illustration of an optical systemof the reflection type according to a first embodiment. This opticalsystem is preferably capable of exciting and measuring fluorescence onor in samples treated using fluorescent pigments and includes a firstlaser 1. This laser emits light of a first wavelength 2—to excite firstfluorescent pigments—so that these first fluorescent pigments emit lightof a second wavelength 3. A mirror 4 deflects the light of the firstwavelength 2, which comes out of the first laser 1 and is incident onthe mirror 4 parallel to an optical axis 5, in the direction of a sample6 (cf. FIGS. 2 and 3). A deflection element 7 deflects the light 2 fromthe first laser 1 onto this mirror 4. An optic 8 produces a first focalpoint 9 for the incident light from the first laser 1 deflected by themirror 4 of the first wavelength 2. A unit 10 includes the mirror 4 andthe optic 8. This mirror 4 and the optic 8 are positioned fixed inrelation to one another in the unit 10.

This unit 10 is positioned so it is movable back and forth linearlyalong the optical axis 5 and is mechanically connected to an oscillatinglinear drive 11. The oscillating linear drive 11 may be implemented, forexample, as a stack of piezoelements; as a mechanical drive, including aconnecting rod, for example, or as a naturally oscillating solid bodyexcited by ultrasound waves. However, the implementation of anoscillating linear drive 11 as a “voice coil”, as is described in U.S.Pat. No. 5,260,569 and particularly in U.S. Pat. No. 5,880,465, ispreferred. In this case, a device is used to produce the back and forthmovement which essentially corresponds to a loudspeaker, the membranemovements being transferred to the unit 10 using a mechanicalconnection.

This optic 8 is implemented accordingly, so that it additionally acts asa collimator for the light of second wavelength 3 emitted by the firstfluorescent pigments (“light originating from the sample”). The mirror 4additionally deflects this collimated light 12 diametrically opposite tothe direction of incidence of the light of first wavelength 3 andparallel to the optical axis 5.

This optical system additionally includes a table 13, movable at leastin the direction of the X and Z spatial axes, for samples 6 treatedusing at least one first fluorescent pigment and for aligning the sample6 in relation to the first focal point 9. The table 13 is preferablyadditionally movable in the direction of the Y spatial axis, the X axisand the Y axis running at least approximately parallel to the surface 15of a sample holder 14.

The X, Y, and Z spatial axes are at least approximately perpendicular toone another, the Y axis extending parallel to a horizontally runningpart of the optical axis 5 and to the geometrical axis G, which alsoruns horizontally.

The table 13 is preferably constructed in multiple parts and includes apart 13′, movable in the direction of the X axis over a smaller distance(thin arrow), a part 13″, movable in the direction of the X axis over alarger distance (thicker double arrow), and a part 13′″, movable in thedirection of the Y axis over a larger distance (thicker double arrow).The entire table 13 or at least the part 13′ is movable in the directionof the Z axis. Electric motors are preferably used for moving the partsof the table 13.

Slides made of glass, for example, as have been known from lightmicroscopy for a long time, are suitable as sample holders 14. Otherand/or similar sample holders may be produced from plastic. Still other,essentially flat sample holders, which are also suitable for scanningtunnelling microscopy, for example, may be implemented from silicon orpyrolytic graphite and the like and/or include these materials. Sampleholders may also be used which have a defined partitioning on thesurface. This partitioning may include a uniform, grooved division;however, it may also have an array of depressions. Examples of sampleholders having such an array of depressions are silicon or glass plateshaving multiple etched depressions. Further examples are standardmicrotitration plates (trademark of Beckman Coulter, Inc., 4300 N.Harbour Blvd., P.O. Box 3100 Fullerton, Calif., USA 92834) or“microplates”, which include 96, 384, 1536, or more depressions in theform of “wells”.

While dry or at least partially dried or immobilized samples arepreferably prepared for investigation on slides, sample holder 14 havingdepressions and/or wells may accommodate liquid samples 6 or samples 6located in a liquid. A frame 31 which essentially has the externaldimensions of a microplate has been shown to be especially suitable foruse in the optical system according to the present invention. This frametherefore fits in and/or on the same sample table 13 as a microplate andmay also be placed there or removed therefrom automatically like amicroplate, i.e., using a robot arm. This frame 31 is implemented forreceiving and holding multiple slides, particularly made of plastic,glass, silicon, pyrolytic graphite, and the like. The arrangement of 4glass slides on such a frame has especially proven itself. In this case,the object carriers 14 are arranged, as shown in FIG. 1, for example, ina row, long edge to long edge—each pushed together or at a slightdistance to one another (cf. FIG. 1). The frame 31 is preferablyproduced from plastic in the injection molding method, through which theproduction price may be kept low and the dimensional precision may bekept high. This dimensional precision makes using such frames 31 in allpossible devices for automated handling of microplates significantlyeasier. Of course, the frames 31 may also be equipped with the slides 14and inserted into the optical system according to the present inventionby hand. Frames 31 equipped with slides 14 additionally ease the furtherhandling of slides secured in this way, which now no longer have to begrasped directly between the process steps to be performed in thedevices for automated handling.

The optical system additionally includes an optical arrangement 16 forimaging a second focal point 17 using the light of the second wavelength3 emitted by the first fluorescent pigments, collimated by the optic 8,and deflected by the mirror 4 (“light originating from the sample”). Anaperture plate 18 positioned in the second focal point 17 is used formasking light of the second wavelength 3 which meets this aperture plate18 at a distance greater than a specific distance from the focal point17. This aperture plate 18 is preferably implemented as replaceable, sothat using multiple diameters of this confocal aperture plate, thesharpness and/or the brightness of the second focal point may beincreased or reduced as required. Especially in the event of extremelypoor light conditions, a larger plate, which causes a reduction of theimaging sharpness, may be useful. A replaceable aperture plate 18 allowsselection of different aperture diameters for adjusting the imaging inthe second focal point 17 to the sample volume and/or to the desiredpenetration depth of the excitation beam into the sample and selectionof the corresponding depth of field of the detector arrangement.

The optical system also includes a first spectral filter 19 forselecting a component of the light of the second wavelength 3 passingthrough the aperture plate 18 and a first detector 20 for measuring theintensity of the light of the second wavelength 3 transmitted throughthe aperture plate 18 and selected by the first spectral filter 19(“light originating from the sample”).

The optical system according to the present invention includes acontinuous geometrical axis G, which does not run through the sample, onwhich the optical arrangement 16, the second focal point 17, theaperture plate 18, the first spectral filter 19, and the first detector20 are positioned—together with the mirror 4 and the deflection element7—this geometrical axis G being at least partially identical to theoptical axis 5 in the region between the mirror 4 and the first detector20.

This joint arrangement on the geometrical axis G has the advantage thatthe scanning of the sample may be performed at high speed using a verysimply constructed unit 10 movable back and forth. This unit 10 actuallyonly includes the mirror 4 and the optic 8. The oscillation frequency ispreferably 20 Hz, the oscillation amplitude D able to be up to 25 mm ormore.

The light of the laser 1 placed distal from this unit 10 and also thelight, originating from the sample, running parallel and diametricallyopposite to that of the laser 1, represents a purely optical connectionbetween the scanning part of the system and the excitation andmeasurement part of the system, so that no oscillations are transferredfrom the scanning or raster part to the measurement part of the system.In addition, this arrangement allows the bundled light 2 of the laser 1to be fed directly into the optical system. The mirror 4 deflects thebundled laser beam 2 onto the optic 8, which in turn always deflects theresulting focal point onto the same point on the optical axis 5. Thisoptical axis 5 preferably runs vertically between the mirror 4 and thesample 6. This optical system, based on coupling a bundled laser beam,is subject to fewer light losses than those which feed and deflect acollimated light beam in order to obtain a first focal point.

In the system according to the present invention, the optical axis 5 isidentical to the geometrical axis G along a part which preferably runshorizontally. In a direction parallel to these axes 5,G, a bundled laserbeam is used to excite the sample, and a collimated beam having lightoriginating from the sample is used in the direction diametricallyopposite thereto. In this way, the deflection element 7, which is todiffract the laser beam 2 in the direction of the mirror 4, but which isto be transparent to the collimated light beam, may be a simple glassplate. Typically, approximately 4% of a light beam is reflected andapproximately 96% is transmitted at a boundary surface glass/air.Antireflection coating increases the transmission if necessary andreduces the reflection of such a glass plate. This simple glass plate ordisk replaces the more expensive polarization beam splitter known fromU.S. Pat. No. 5,260,569 (see FIG. 1 therein, reference number 13) andquarter-wave plates (see FIG. 1 therein, reference number 18), which arenecessary in the related art because a collimated light beam is used inboth directions, each of which requires the entire surface of the mirrorand filter.

A further advantage of the system according to the present invention isthat the laser beam 2 for exciting the sample fluorescence must runparallel to the optical axis 5, but not in its center and not in thecenter of the geometrical axis G. This is advantageous above all fordetermining a mutually optimum Z position of sample 6 and first focalpoint 9. The laser beam then preferably runs at a distance (A) to theoptical axis 5, which is identical to the geometrical axis G. Therefore,“off-center illumination” of the sample is possible, as is shown inFIGS. 2 and 3, for example.

Furthermore, the system according to the present invention includes thepossibility of using a preferred deflection element 7, which, fordeflecting the light 2 from the first laser 1 onto this mirror 4 in adirection parallel to the optical axis 5, includes a highly reflectiveregion 21 intended for the laser beam 2 and is essentially transparentin its remaining regions to the light of both the first wavelength 2 andthe second wavelength 3. Therefore, if such a “pin mirror” is used, agreatly improved yield for the bundled laser light for exciting thefluorescence of the fluorescent pigments in or on the samples 6 resultsin comparison to the simple glass plate used as the deflection element7. The loss of approximately 5% of the light originating from the sampleand deflected on the highly reflective region 21 is not of greatimportance here.

In order to make the device more compact, the optical system mayadditionally include a simple mirror for reflecting the light beamsrunning between the deflection element 7 and the mirror 4 (not shown).This simple mirror is then also positioned in the geometrical axis (G).However, in this case the optical arrangement 16, the second focal point17, the aperture plate 18, the first spectral filter 19, and the firstdetector 20—corresponding to the deflection by the simple mirror—arepositioned on a geometric axis G′, which is different from G.

The optical system shown in FIG. 1, having two preferably monochromaticlasers of different wavelengths, additionally includes a second laser1′, which—to excite second fluorescent pigments—emits light of a thirdwavelength 2′ running parallel to the light of the first wavelength 2,so that these fluorescent pigments emit light of a fourth wavelength 3′.A second spectral filter 19′ selects a component of the light of thefourth wavelength 3′ and a second detector 20′ measures the intensity ofthe part of the light of the fourth wavelength 3′ selected by the secondspectral filter 19′. In addition, a beam splitter element 26 isprovided, which is at least partially transparent to the light 12 of thesecond wavelength 3 emitted by the first fluorescent pigments andcollimated by the optic 8 and which is implemented for reflecting and/ordeflecting the light of the fourth wavelength 3′ from the geometric axisG in the direction of a second detector 20′. This beam splitter element26 may be designed as a dichroic mirror or even, for example, as a 50%beam splitter.

An object illuminator 30 in the form of a simple lamp, for example, ispreferably also provided distal from the unit 10, so that the objects tobe scanned may be observed and/or imaged using light optics ifnecessary. The light used is fed using a dichroic mirror positioned onthe axes 5, G. The spectral filters 19, 19′ are designed in such a waythat they filter out this light. A separate detector (not shown) may beprovided in order to capture the visual image of the samples 6.Alternatively, the detector 20 may also be used in order to scan thesamples in the fluorescence mode, for example.

FIG. 2 shows a vertical partial section of an optical system forexciting and measuring fluorescence on or in samples treated usingfluorescent pigments according to a second embodiment. This opticalsystem is claimed in at least one of the attached claims. For thissecond, somewhat simpler embodiment, only one monochromatic laser 1 isprovided. The table 13 is additionally movable in the direction of the Yspatial axis, X and Y axes running at least approximately parallel tothe surface 15 of a sample holder 14. The sample holders described inFIG. 1 may all also be used in this embodiment.

The deflection element 7, for deflecting the light 2 from the firstlaser 1 onto this mirror 4 in a direction parallel to the optical axis5, includes a highly reflective region 21 intended for the laser beam 2.In its remaining regions, the deflection element 7 is essentiallytransparent to the light of both the first wavelength 2 and the secondwavelength 3. The highly reflective region 21 of the deflection element7 is positioned at a distance A to the optical axis 5, which isidentical to the geometrical axis G, so that the beam path for the lightof the first wavelength 2 coming out of the laser 1 runs parallel and ata distance A to these axes 5, G.

An opaque screen 22 is positioned between the deflection element 7 andthe optical arrangement 16 for intermittent masking of a laser beam,used for focusing and excitation and reflected by the sample 6. In thestate shown, this screen 22 is pushed into the beam path.

The bundled light 2 coming from the laser 1 is deflected on the highlyreflective region 21 of the deflection element 7 in the direction of themirror 4—preferably slanted at 45° to the horizontal—parallel to theaxes 5, G and at a distance A to these axes. The mirror 4 deflects thisbundled laser beam 2 parallel and at the distance A to the now at leastapproximately vertically running optical axis 5, upon which the laserbeam is deflected into the focal point 9 lying in the optical axis 5.The previously described course of the laser beam 2 is used forfocusing, i.e., for fixing an optimum operating distance between optic 8and sample 6. At the same time, the mutual position of focal point 9 andsample 6 is determined and set (as described below). The focusing beam25 concentrated on the focal point 9 has a spot diameter between thediffraction limit and 2 mm. A preferred spot diameter is less than 15μm. The focusing beam 25 is reflected by the sample and, at a distancefrom the optical axis 5, reaches the mirror 4 again, which deflects thisreflected beam in the direction parallel to the horizontally runningaxes 5, G.

This off-center illumination is especially suitable for determining anexact operating distance—which is preferably up to 7 mm, but may also bemore or less—because the laser beam is incident on the sample at anangle less than 90°. It is obvious that as the angle of incidence isreduced, the precision and/or the resolution in the direction of the Zaxis increases, while an angle of incidence of 90° allows the lowestresolution in the direction of the Z axis, because under certaincircumstances it may not be determined, for example, at what depth thefocal point 9 is located inside the sample 6. In addition to this, theresolution of the optical system in the direction of the Z axisincreases with increasing distance A. The reflected beam runningparallel to the axes 5, G is deflected by an optical arrangement 16 intothe second focal point 17, where it passes through the aperture plate18. The spectral filter is now preferably pulled out of the beam path orreplaced by a neutral density filter (gray filter), so that thereflected beam is incident on the detector 20 and the detector maymeasure the intensity of the reflected beam.

If the optimum operating distance is not to be set using the reflection,but rather on the basis of the intensity of the fluorescence excited inthe sample(s) and/or on the basis of diffuse scattered light generatedin or on the sample 6, the screen 22 is pushed into the beam path (cf.FIG. 2) and the spectral filter 19 and/or a neutral density filter (grayfilter) is placed in front of the detector 20. The bundled laser beam 1is then used as the excitation beam 24, which is deflected into thefirst focal point 9 in the same way as the focusing beam 25. A part ofthe fluorescence, which propagates essentially in a dome shape from thesample 6, is collimated (i.e., aligned in parallel) in the optic 8 anddeflected by the mirror 4 in the direction of the optical arrangement16. In spite of a small loss, which is essentially determined by thearea of the highly reflective region 21 of the deflection element 7 andthe screen 22, this collimated fluorescent light is incident at theoptical arrangement 16 and is focused in the second focal point 17.There—depending on the diameter of the aperture plate 18 selected—acertain part of the focused fluorescent light passes through thisaperture plate 18 and the spectral filter 19 and is detected by thedetector 20. The same beam path as just described is used for scanningthe sample.

The first spectral filter 19 is positioned between the aperture plate 18and the first detector 20. It may also be used as a window in thedetector 20. However, the implementation of the first spectral filter 19as a filter slider having, for example, five different filters which maybe automatically replaced with one another is preferred, this spectralfilter 19 being manually replaceable by another filter slider havinganother filter set.

This optical system preferably includes a computer or microprocessor forrecording and processing the measurement signals detected by thedetector 20 and for outputting data corresponding to these signals. Thiscomputer or microprocessor is preferably also implemented forcontrolling the movements of the table 13. The table 13, which isdisplaceable in the directions of the X and/or Y and/or Z axes, ispreferably also implemented so it may be tilted around the X and/or Yaxes.

According to the method according to the present invention, such anoptical system is provided with a continuous geometrical axis G, whichdoes not run through the sample. The optical arrangement 16, the secondfocal point 17, the aperture plate 18, the first spectral filter 19, andthe first detector 20—together with the mirror 4 and the deflectionelement 7—are positioned on this geometrical axis G. In this case, thisgeometrical axis G is at least partially identical to the optical axis 5in the region between the mirror 4 and the first detector 20.

During the excitation and measurement of the fluorescence emitted by thesample 6, the unit 10 of this optical system is fixed according to afirst type of use. The sample table 13 is simultaneously moved in thedirection of the X and/or Y axes, the Y axis running parallel to theaxes 5, G. Using this table movement, which may be performed by theparts 13′ (X movement) and 13′″ (Y movement), for example, a linear scanon or in the samples 6 is made possible with continuous excitation andmeasurement, and a point scan is made possible with point excitation andpoint measurement.

During the excitation and measurement of the fluorescence emitted by thesample 6, the unit 10 of this optical system is moved back and forth inthe direction of the Y axis according to a second type of use. Thesample table 13 is simultaneously fixed and/or moved in the direction ofthe X axis. Using only these movements of the unit 10, a linear scan onor in the samples 6 is made possible with continuous excitation andmeasurement, and with simultaneous movement of unit 10 (Y movement) andtable 13 (movement of the part 13′ results in X movement) an area scanis made possible with continuous excitation and measurement. The Y axisruns parallel to the axes 5, G in this case. The oscillating lineardrive 11 for the unit 10 is preferably implemented as a “voice coil”.

Before the effective scanning (measurement) of the samples, the Zposition of the sample holder is set.

Laser pulses or lamp flashes may be used as an alternative light sourcefor the excitation of the fluorescence. Discrete, individual laserpulses or discrete short series of a few laser pulses are thenpreferred, which excite the fluorescence at one point at a time in or onsamples 6. Such point measurements are preferably performed especiallyduring measurement of the fluorescence in samples provided in the 1536wells of a high-density microplate, for example. Between or during themeasurements, unit 10 and sample holder 14 are displaced in relation toone another in this case, the displacement of the unit 10 in thedirection of the Y axis above all able to be performed extremelyrapidly. The measurement of the fluorescence in these 1536 wells, whichlasts approximately 1 minute in area scan with continuous excitation andmeasurement using the most rapid current devices, may thus be shortenedto approximately 10 seconds. For microplates having even more wells oran even smaller scanning dimension, an even larger time savings results.

For samples 6 immobilized on essentially flat sample holders 14, forexample, the opaque screen 22, which is positioned between thedeflection element 7 and the optical arrangement 16, is pulled out ofthe beam path to define an optimum Z position of the movable table 13and/or a sample 6. Subsequently—on the basis of a series of measurementsignals generated during a Z movement of the table 13 by the firstdetector 20 and recorded in a computer or microprocessor—the Z positionof the table corresponding to the maximum of these measurement signalsis calculated and the table 13 is moved into this Z position. Thegeometric center between the points of inflection of the rising andfalling slopes of the measurement signal is preferably taken to fix themaximum of these measurement signals. This method is preferablyperformed at at least three points of the sample holder and the table 13is displaced—in accordance with the defined maxima for these threepoints—in the directions of the X, Y, and Z axes and tilted around the Xand Y axes as far as necessary. It is advantageous if this definition ofan optimum Z position is performed using a computer or microprocessor,which detects and processes the measurement signals generated by thedetector 20, outputs data corresponding to these signals, and alsocontrols the movements of the table 13.

FIG. 3 shows a vertical partial section of an optical system forexciting and measuring fluorescence on or in samples treated usingfluorescent pigments according to a third embodiment. This opticalsystem is claimed in at least one of the attached claims. One or morepreferably monochromatic lasers 1, 1′ may be provided for this thirdembodiment. The table 13 is preferably movable in the direction of theX, Y, and Z spatial axes, X and Y axes running at least approximatelyparallel to the surface 15 of a sample holder 14. In addition, the table13 may preferably be tilted around the X and/or Y axes. The sampleholders described in FIGS. 1 and 2 may all also be used in thisembodiment.

The deflection element 7 for deflecting the light 2 from the first laser1 onto this mirror 4 in a direction parallel to the optical axis 5includes a highly reflective region 21 intended for the laser beam 2. Inits remaining regions, the deflection element 7 is essentiallytransparent to the light of both the first wavelength 2 and the secondwavelength 3. The highly reflective region 21 of the deflection element7 is positioned in the center of the optical axis 5, which is identicalto the geometrical axis G, so that the beam path for the light of thefirst wavelength 2 coming out of the laser 1 runs parallel to and in thecenter of these axes 5, G. This optical system includes a separatingelement 23 for separating the light of the first wavelength 2 coming outof the laser 1 into an excitation beam 24 for the fluorescence and afocusing beam 25 parallel to this excitation beam 24. An opaque screen22 is positioned between the deflection element 7 and the separatingelement 23 for intermittent masking of a laser beam used to excite thefluorescence. This separating element 23 may be implemented as a simple,plane-parallel glass plate having complete mirroring on the back, ifonly one laser 1 is to be used.

The bundled light 2 from the laser 1 is reflected approximately 4% inthe direction of the deflection element 7 on the non-mirrored firstsurface of the separating element 23. There, this laser beam is incidenton a non-mirrored region and is again reflected approximately 4% and ata distance A parallel to the axes 5, G in the direction of the mirror 4.The bundled light 2 coming from the laser 1 is additionally reflected onthe rear surface of the separating element 23. With a glass platecompletely mirrored on the back, approximately 96% of the laser light 2on optical axis 5 is deflected on the deflection element 7, where thelight 2 is incident on its highly reflective region 21. The separatingelement 23 thus fulfills the object of separating the laser beam 2 intoan excitation beam 24 running on the optical axis 5 and a focusing beam25 running parallel thereto at a distance A.

A simple, plane-parallel glass plate which is placed diagonally in thebeam path may also be used as an alternative separating element. Theposition of this plane-parallel glass plate is to be located between thelaser 1 and the deflection element 7; however, this plane-parallel glassplate is preferably positioned between the laser 1 and the separatingelement 23 (not shown). The largest component of the laser beam passesthrough the glass plate with a slight parallel offset. In this case,approximately 4% is reflected back into the glass plate at the rearboundary surface glass/air. A component of approximately 4% of thereflected beam then subsequently experiences another reflection at thefront boundary surface. This leads to a small component of the laserlight being deflected parallel to the main beam in the direction of thedeflection element 7. In this case, the separating element 23 isimplemented as a front surface mirror and is not used to separate thebeam.

If two or more monochromatic lasers of different wavelengths are to beused, a plane-parallel plate implemented as a dichroic mirror is used asthe separating element 23. Each laser may then—in accordance with thewavelength of its light—be assigned its own separating element 23, 23′,which is transparent to the remaining lasers. These separating elementsare then preferably positioned on a shared optical axis and deflect thelaser beams in the direction of one single deflection element 7, as maybe seen from FIG. 1.

The bundled light 2, 2′ coming from the lasers 1, 1′ is deflected on thehighly reflective region 21 of the deflection element 7 in the directionof the mirror 4—preferably slanted at 45° to the horizontal.

The stronger of the two beams produced by the separating element 23 istherefore preferably incident on the highly reflective region 21 of thedeflection element 7, where it is deflected, in the direction of theoptical axis 5 and the geometrical axis G, identical thereto, onto themirror 4. This excitation beam 24, which is preferably masked forfocusing using inserted screen 22, is deflected by mirror 4 in thedirection of the at least approximately vertically running optical axis5, passes through the optic 8, and is incident on the sample 6 in theoptical axis 5. The excitation beam 24, which is incident on the sample6 at least approximately vertically, is especially suitable for excitingthe fluorescence in samples 6 which are provided immersed in a liquid orin liquid form. Such samples are preferably provided in the wells ofmicroplates for investigation. Even if high-density microplates havingnot only 96, but 384, 1536, or more wells are to be used, the at leastapproximately vertical excitation beam always reaches the samples 6.

In contrast to the use of known and expensive, so-called “f(θ)-optics”for achieving an excitation beam which is incident on a sample 6 atleast nearly perpendicularly, the present invention allows the use ofsignificantly more cost-effective optical elements for the mirror 4 andthe optic 8.

The second, preferably weaker light beam is thus incident on anon-mirrored region of the deflection element 7 where it is(approximately 4%) deflected, running parallel to the direction of theaxes 5, G and at the distance A thereto, onto the mirror 4. Thisfocusing beam 25 is preferably only active when screen 22 is insertedand is deflected by mirror 4 parallel and at a distance A to thedirection of the at least approximately vertically running optical axis5. The optic 8 deflects the focusing beam onto the focal point 9 lyingin the at least approximately vertical optical axis 5.

The considerations of the use of the focusing beam (cf. secondembodiment, FIG. 2) and of the focusing on the basis of the reflection,i.e., for fixing an optimum operating distance between optic 8 andsample 6, also essentially apply here. This is also true for focusingusing measurement of the fluorescence and/or on the basis of diffusescattered light generated in or on the sample 6, the screen 22 remainingpulled out of the beam path if the third embodiment described here isused and the fluorescence is measured.

A part of the fluorescence propagating essentially in a dome shape fromthe sample 6 is collimated (i.e., aligned in parallel) in the optic 8and deflected by the mirror 4 in the direction of the opticalarrangement 16. In spite of a small loss, which is essentiallydetermined by the area of the highly reflective region 21 of thedeflection element 7, this collimated fluorescent light is incident atthe optical arrangement 16 and is focused in the second focal point 17.There, a certain part of the focused fluorescent light—depending on theselected diameter of the aperture plate 18—passes through this apertureplate 18 and the spectral filter 19 and is detected by the detector 20.The same beam path as just described is used for scanning the sample.

The excitation beam, which always runs in the optical axis 5 here, isreflected by the sample 6 and runs on the diametrically opposite path inthe direction of the lasers 1, 1′.

If two monochromatic lasers 1, 1′ are used, a beam splitter element 26is preferably positioned between the aperture plate 18 and the firstdetector 20 (cf. FIG. 3). A first convergent lens 28 is used forcollecting the light of the second wavelength 3 passing through theaperture plate 18. A second convergent lens 28′ is positioned downstreamfrom the beam splitter element 26, using which the light of the fourthwavelength 3′ passing through the aperture plate 18 is collected andsupplied to the second detector 20′ having the second spectral filter19′.

If necessary, further optical elements (lenses, mirrors, screens) may beplaced between the aperture plate 18 and the detector 20—preferablybetween the spectral filter 19 and the detector 20—for optimized beamguiding in regard to signal yield and filter effect.

The reference numbers in the different figures each refer to identicalfeatures. Any arbitrary combinations of the embodiment shown and/ordescribed are included in the scope of the present invention.

Advantages which differentiate the present invention from the relatedprior art include:

-   -   the use of sample holders 14 the size of a microplate or even        larger sample holders is made possible;    -   the operating distance may be up to 7 mm or more;    -   the numerical aperture of the objective is above a value of 0.4        and is up to 0.6 or more;    -   the oscillation amplitude D may be up to 25 mm or more, in        particular thanks to the use of a counter-oscillator 29;    -   the at least approximately vertical excitation of the samples is        achieved using a cost-effective and largely aberration-free        optic;    -   the deflection element 7 may be implemented as a simple glass        disk or as a pin mirror and allows the separation of excitation        beam and fluorescence as a simple optical element;    -   the use of a deflection element 7 in the form of a pin mirror        allows the simultaneous use of multiple monochromatic lasers of        differing wavelengths and the measurement of a nearly unlimited        number of different fluorescent pigments of differing emission        wavelengths;    -   the exchange of two monochromatic lasers of different        wavelengths does not require any further modification of the        optical system;    -   the use of the different focusing modes disclosed in combination        with an aperture plate of different aperture diameter allows        greatly varying samples and sample formats to be brought into        focus, excited, and their fluorescence measured.

1. An optical system, for exciting and measuring fluorescence on or insamples treated using fluorescent pigments, including: at least onefirst laser which emits light of a first wavelength to excite firstfluorescent pigments, so that these first fluorescent pigments emitlight of a second wavelength; a mirror for deflecting the bundled lightof the first wavelength, which comes out of the first laser and isincident on the mirror parallel to an optical axis, in the direction ofa sample; deflection element for deflecting the bundled light from thefirst laser onto this mirror; an optic, for producing a first focalpoint for the bundled light of the first wavelength, which is incidentfrom the first laser and is deflected by the mirror; a unit, whichincludes the mirror and optic, the mirror and optic being positionedfixed in relation to one another in the unit and this unit beingpositioned so it is linearly movable back and forth along the opticalaxis and being mechanically connected to an oscillating linear drive,the optic additionally being implemented as a collimator for the lightof the second wavelength emitted by the first fluorescent pigment andthe mirror additionally being implemented for deflecting this collimatedlight diametrically opposite to the direction of incidence of thebundled light of the first wavelength and parallel to the optical axis;a table, movable at least in the direction of the X and Z spatial axes,for receiving sample holders for samples treated using at least onefirst fluorescent pigment and for aligning the sample in relation to thefirst focal point; an optical arrangement for imaging a second focalpoint using the light of the second wavelength, which is emitted by thefirst fluorescent pigment, collimated by the optic and deflected by themirror; an aperture plate, positioned in the second focal point, formasking light of the second wavelength, which is incident on thisaperture plate at a distance from the focal point greater than aspecific distance; a first spectral filter for selecting a component ofthe light of the second wavelength passing through the aperture plate;and a first detector for measuring the intensity of the light of thesecond wavelength which passes through the aperture plate and isselected by the first spectral filter, wherein the optical systemincludes a continuous geometrical axis (G), which does not run throughthe sample, on which the optical arrangement, the second focal point,the aperture plate, the first spectral filter, and the firstdetector—together with the mirror and the deflector element—arepositioned, this geometrical axis (G) being at least partially identicalto the optical axis in the region between the mirror and the firstdetector, wherein the deflection element includes a highly reflectivezone, deflecting the bundled light from the first laser onto this mirrorin a direction parallel to the optical axis, and wherein the deflectionelement in its remaining regions is essentially transparent to the lightof both the first wavelength and the second wavelength.
 2. The systemaccording to claim 1, wherein the table is additionally movable in thedirection of the Y spatial axis, X and Y axes lying at leastapproximately parallel to the surface of the sample holder.
 3. Thesystem according claim 1, wherein the highly reflective region of thedeflection element is positioned at a distance (A) to the optical axis,which is identical to the geometrical axis (G), so that the beam pathfor the bundled light of the first wavelength coming out of the laserruns parallel and at a distance (A) to these axes.
 4. The systemaccording to claim 3, wherein an opaque screen—for intermittent maskingof a laser beam used for focusing and excitation and reflected from thesample—is retractably positioned between the deflection element and theoptical arrangement.
 5. The system according to claim 1, wherein thesystem additionally includes a separating element for separating thelight of the first wavelength coming out of the laser into an excitationbeam and a focusing beam, which is parallel to this excitation beam. 6.The system according to claim 5, wherein an opaque—screen forintermittent masking of a laser beam used for exciting the fluorescentpigments—is retractably positioned between the deflection element andthe separating element.
 7. The system according to claim 1,characterized in that the first spectral filter is positioned betweenthe aperture plate and the first detector.
 8. The system according toclaim 1, which includes: a second laser, which—to excite secondfluorescent pigments—emits light of a third wavelength running parallelto the light of the first wavelength, so that these fluorescent pigmentsemit light of a fourth wavelength; a second spectral filter forselecting a component of the light of the fourth wavelength; and asecond detector for measuring the intensity of the part of the light ofthe fourth wavelength selected by the second spectral filter, whereinthe system additionally includes a beam splitter element, which is atleast partially transparent to the light of the second wavelengthemitted by the first fluorescent pigments and collimated by the optic,and which is implemented for reflecting and/or deflecting the light ofthe fourth wavelength from the geometrical axis (G) in the direction ofa second detector.
 9. The system according to claim 1, wherein thesystem additionally includes at least one convergent lens for collectingthe light of the second wavelength passing through the aperture plate.10. The system according to claim 8, wherein the system additionallyincludes at least one convergent lens for collecting the light of thesecond wavelength and/or fourth wavelength passing through the apertureplate.
 11. The system according to claim 8, characterized in that thebeam splitter element is positioned between the aperture plate and thefirst detector.
 12. The system according to claim 1, wherein the sampleholder which may be received by the sample table is implemented as aslide, particularly made of plastic, glass, silicon, or pyrolyticgraphite.
 13. The system according to claim 1, wherein the sample holderwhich may be received by the sample table is implemented as amicroplate, particularly having 96, 384, 1536 , or more wells.
 14. Thesystem according to claim 1, wherein the sample holder which may bereceived by the sample table is implemented as a frame for receiving andholding multiple slides, particularly made of plastic, glass, silicon,or pyrolytic graphite, and has the external dimensions of a microplate.15. The system according to claim 1, wherein the unit —during theexcitation and measurement of the fluorescence emitted by thesamples—may be fixed and the sample table is implemented assimultaneously movable in the direction of the X and/or Y axes, the Yaxis running parallel to the geometrical axis (G).
 16. The systemaccording to claim 1, wherein the sample table—for excitation andmeasurement of the fluorescence emitted by the samples —is implementedas movable in the direction of the X axis and the unit is simultaneouslymovable back and forth in the direction of the Y axis, the Y axisrunning parallel to the geometrical axis (G).
 17. The system accordingto claim 16, wherein the oscillating linear drive is implemented as avoice coil.
 18. The system according to claim 16, wherein the unit alsoincludes a counter-oscillator which—to absorb oscillations of theunit—is movable counter to the linear drive in the direction of the Yaxis.
 19. The system according to claim 1, wherein the aperture plate isimplemented as replaceable for various aperture sizes.
 20. The systemaccording to claim 8, wherein the first spectral filter is implementedas a filter slider having at least two different filters which may beautomatically replaced with one another and this filter slider isimplemented so it may be manually replaced by another filter slider. 21.The system according to claim 8, wherein the beam splitter element isimplemented as a carriage slider having replaceable mirrors.
 22. Thesystem according to claim 1, wherein the system includes a computer ormicroprocessor for recording and processing the measurement signalsdetected by detectors and for outputting data corresponding to thesesignals.
 23. The system according to claim 22, wherein the computer ormicroprocessor is additionally implemented for controlling the movementsof the table, the table being implemented as displaceable in thedirections of the X and/or Y and/or Z axes and tiltable around the Xand/or Y axes.
 24. A method of exciting and measuring fluorescence on orin samples treated using fluorescent pigments using an optical system,in which at least: a first laser, which—to excite these fluorescentpigments—emits light of a first wavelength, so that these fluorescentpigments emit light of a second wavelength; a mirror, for deflecting thebundled light of the first wavelength, coming out of the first laser andincident on the mirror parallel to an optical axis, in the direction ofa sample; a deflection element for deflecting the bundled light from thefirst laser onto this mirror; an optic, for producing a first focalpoint for the bundled light of the first wavelength, which is incidentfrom the first laser and is deflected by the mirror; a unit, whichincludes the mirror and the optic, mirror and optic being positionedfixed in relation to one another in the unit and this unit beingpositioned so it is movable back and forth along the optical axis andbeing mechanically connected to an oscillating linear drive, the opticadditionally being implemented as a collimator for the light of thesecond wavelength emitted by the fluorescent pigments and the mirroradditionally being implemented for deflecting this collimated lightdiametrically opposite to the direction of incidence of the bundledlight of the first wavelength and parallel to the optical axis; a table,movable at least in the direction of the X and Z spatial axes, forreceiving sample holders for samples treated using at least one firstfluorescent pigment, the X axis running at least approximately parallelto the surface of the sample holder, which—for aligning the sample inrelation to the first focal point—is movable using the table in thedirection of the Z axis; an optical arrangement for imaging a secondfocal point using the light of the second wavelength, which is emittedby the fluorescent pigments, collimated by the optic, and deflected bythe mirror; an aperture plate, positioned in the second focal point, formasking light of the second wavelength, which is incident on thisaperture plate at a distance from the focal point greater than aspecific distance; a first spectral filter for selecting a component ofthe light of the second wavelength passing through the aperture plate;and a first detector for measuring the intensity of the light of thesecond wavelength transmitted by the aperture plate and selected by thefirst spectral filter, are used, wherein the method comprises theprovision of an optical system having a continuous geometrical axis (G),which does not run through the sample, and on which the opticalarrangement, the second focal point, the aperture plate, the firstspectral filter, and the first detector—together with the mirror and thedeflection element—are positioned, this geometrical axis (G) being atleast partially identical to the optical axis in the region between themirror and the first detector, and wherein with the deflection elementthat includes a highly reflective region intended for the laser beam andthat is essentially transparent to the light of both the firstwavelength and the second wavelength in its remaining regions, bundledlight from the first laser is deflected onto this mirror in a directionparallel to the optical axis.
 25. The method according to claim 24,wherein the highly reflective region of the deflection element ispositioned at a distance (A) to the optical axis, which is identical tothe geometrical axis (G), so that the beam path for the bundled light ofthe first wavelength coming out of the laser is made running paralleland at the distance (A) to this axis (G).
 26. The method according toclaim 24, wherein—to define an optimum Z position of the movable tableand/or a sample—an opaque screen, positioned between the deflectionelement and the optical arrangement, is pulled out of the beam path andwherein—on the basis of a series of measurement signals generated duringa Z movement of the table by the first detector and recorded in acomputer or microprocessor—the Z position of the table corresponding tothe maximum of these measurement signals is calculated and the table ismoved into this Z position.
 27. The method according to claim 24,wherein—to define an optimum Z position of the movable table and/or asample—an opaque screen, positioned between the deflection element andthe separating element is pushed into the beam path, and wherein—on thebasis of a series of measurement signals generated during a Z movementof the table by the first detector and recorded in a computer ormicroprocessor—the Z position of the table corresponding to the maximumof these measurement signals is calculated and the table is moved tothis Z position.
 28. The method according to claim 26, wherein thegeometrical center between the points of inflection of the rising andfalling slopes of the measurement signal is taken to fix the maximum ofthese measurement signals.
 29. The method according to claim 26, whereinthis method is performed at at least three points of a sample holder andthe table—in accordance with the defined maxima for these threepoints—is displaced as far as necessary in the directions of the X, Yand Z axes and tilted around the X and Y axes.
 30. The method accordingto claim 29, wherein the method is performed using a computer ormicroprocessor, which detects and processes the measurement signalsgenerated by detectors, outputs data corresponding to these signals, andadditionally controls the movements of the table.
 31. The methodaccording to claim 24, wherein the table—before the measurement of thefluorescence on and/or in at least one sample—is displaced in thedirection of the X and/or Y axes and is stopped for the excitation andmeasurement of the fluorescence—while simultaneously fixing the unit.32. The method according to claim 24, wherein the table—during theexcitation and measurement of fluorescence on and/or in at least onesample and while simultaneously fixing the unit—is displaced in thedirection of the X and/or Y axes.
 33. The method according to claim 24,wherein—for exciting and measuring the fluorescence on and/or in atleast one sample—while simultaneously oscillating the unit in thedirection of the Y axis—the table is fixed.
 34. The method according toclaim 24, wherein—for exciting and measuring the fluorescence on and/orin at least one sample—while simultaneously oscillating the unit in thedirection of the Y axis—the table is displaced linearly in the directionof the X axis.
 35. The method according to claim 31, wherein thecorresponding detector signals are recorded and processed in thecomputer or microprocessor.
 36. The method according to claim 31,wherein the opaque screen is pushed into the beam path and/or removedtherefrom.
 37. The method according to claim 31, wherein the excitationand measurement of the fluorescence on and/or in a sample is performedat an optimized Z position of the movable table, sample holder, and/or asample and the corresponding detector signals are recorded and processedin the computer or microprocessor.
 38. The method according to claim 24,wherein only one light flash and/or a few discrete light flashes persample are emitted to excite the fluorescence.
 39. The method accordingto claim 24, wherein multiple lasers are used for generating bundledexcitation light of different wavelengths in or on a multiplefluorescent pigments and multiple detectors are used for measuring thecorresponding signals.
 40. The method according to claim 24, whereinsignals of at least two lasers, fluorescent pigments, and detectors arerecorded, processed and output superimposed.
 41. The method according toclaim 27, wherein the geometrical center between the points ofinflection of the rising and falling slopes of the measurement signal istaken to fix the maximum of these measurement signals.
 42. The methodaccording to claim 27, wherein this method is performed at at leastthree points of a sample holder and the table—in accordance with thedefined maxima for these three points—is displaced as far as necessaryin the directions of the X, Y and Z axes and tilted around the X and Yaxes.
 43. The method according to claim 42, erein the method isperformed using a computer or microprocessor, which detects andprocesses the measurement signals generated by detectors, outputs datacorresponding to these signals, and additionally controls the movementsof the table.